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BI 3: Gene Cloning

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by

Kimberly Christian

on 29 January 2016

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Transcript of BI 3: Gene Cloning

Genetic Engineering:
Bacterial Transformation

Overview
Tools
Procedure
Applications
why engineer bacteria?
simple genome (single chromomsome)
non-essential DNA stored in easily removed and manipulated plasmids
rapidly reproducing organisms
small organisms, easily maintained with minimal requirements
utilize same basic DNA code for proteins as all other organisms
genetic engineering - manipulation of genes for practical purposes (ex: medical research, agriculture)
recombinant DNA - DNA molecule made in the lab using segments of DNA from two different sources
transgenic organism - organism whose genome contains a gene from another organism of the same or different species
recombinant bacteria - bacteria expressing unique phenotypes as a result of the uptake of recombinant plasmid DNA
c. plasmid vector - non-essential bacterial DNA that can be removed from a bacteria, engineered to take up source DNA, and placed back into bacteria
d. restriction enzyme - enzyme isolated from bacteria that cuts up DNA at specific sequences called restriction sites (in nature serve to protect bacteria from viral infection)
sticky ends - single-stranded ends of DNA resulting after cut at restriction site
e. DNA ligase - enzyme that seals fragments of DNA together with phosphodiester linkages
a. source DNA - gene of interest isolated from any organism's genome
b. competent bacteria - bacteria capable of undergoing transformation by taking up plasmids from environment
1. Isolate DNA plasmid from bacteria.
2. Locate source DNA of interest (gene to be inserted into bacteria).
3. Cut both samples of DNA with the SAME restriction enzyme to leave matching sticky ends.
4. Mix cut plasmids, isolated gene of interest, and ligase to seal gene into plasmids.
5. Mix recombinant plasmid with competent bacteria, allowing bacteria to take up plasmids.*
6. Select for replication of transformed bacteria and against non-transformed bacteria.
*Not all bacteria will be competent to take up plasmid - these bacteria will not express the traits associated with the desired gene.
human insulin gene cloning - insulin gene is inserted into bacteria; bacteria multiply and reproduce human insulin protein for use in treating diabetes
Bt corn - bacterial gene is inserted into corn genome; corn produce protein toxin that kills corn borer caterpillers feeding on corn
Do Now:
If you could modify any organism to express a gene from another organism, what would you do?
Referring to the transgenic species you proposed at the start of class, summarize the protocol required to accomplish that genetic engineering. (Don’t just copy the procedure from your notes – your host organism is probably not a bacterium.)
Full transcript